kampo medicine: evaluation of the pharmacological activity of 121 herbal...

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ORIGINAL RESEARCHpublished: 29 July 2016

doi: 10.3389/fphar.2016.00219

Edited by:Adolfo Andrade-Cetto,

National Autonomous Universityof Mexico, Mexico

Reviewed by:Manoj Gajanan Kulkarni,

University of KwaZulu-Natal,South Africa

Wendy A. Stirk,University of KwaZulu-Natal,

South Africa

*Correspondence:Günter Gisselmann

[email protected]

†Present address:Markus Werner, Department of

Biochemistry I – ReceptorBiochemistry, Ruhr UniversityBochum, Bochum, Germany

Specialty section:This article was submitted to

Ethnopharmacology,a section of the journal

Frontiers in Pharmacology

Received: 17 May 2016Accepted: 06 July 2016Published: 29 July 2016

Citation:Hoffmann KM, Herbrechter R,

Ziemba PM, Lepke P, Beltrán L,Hatt H, Werner M and Gisselmann G

(2016) Kampo Medicine: Evaluationof the Pharmacological Activity of 121Herbal Drugs on GABAA and 5-HT3AReceptors. Front. Pharmacol. 7:219.

doi: 10.3389/fphar.2016.00219

Kampo Medicine: Evaluation of thePharmacological Activity of 121Herbal Drugs on GABAA and 5-HT3AReceptorsKatrin M. Hoffmann1, Robin Herbrechter1, Paul M. Ziemba1, Peter Lepke2,Leopoldo Beltrán1, Hanns Hatt1, Markus Werner1† and Günter Gisselmann1*

1 Department of Cell Physiology, Ruhr University Bochum, Bochum, Germany, 2 Kronen Apotheke Wuppertal, Wuppertal,Germany

Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinesemedicine (TCM). During the last several decades, much attention has been paid tothe pharmacological effects of these medical plants and their constituents. However, inmany cases, a systematic screening of Kampo remedies to determine pharmacologicallyrelevant targets is still lacking. In this study, a broad screening of Kampo remedies wasperformed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands.Several of the Kampo remedies are currently used for symptoms such as nausea,emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore,the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzedas ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due tothe involvement of these receptors in such pathophysiological processes. The tincturesof Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effectiveinhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredientsin these compounds led to the identification of leonurine from Leonurus as a newnatural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis(cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identifiedfor the GABAA receptor, which are all traditionally used for their sedative or anxiolyticeffects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were alsodetected. Therefore, this study reveals new insights into the pharmacological action ofa broad spectrum of herbal drugs from Kampo, allowing for a better understanding oftheir physiological effects and clinical applications.

Keywords: Kampo, Leonurus japonicus (herba), Panax ginseng (radix), Salvia miltiorrhiza (radix), andro-grapholide, leonurine, 5-HT3A receptor, GABAA receptor

INTRODUCTION

The 5-HT3A and GABAA receptors are ionotropic receptors within the cys-loop superfamilyof ligand-gated ion channels and therefore possess closely related structures. Both of thesereceptors share a pentameric structure, with each subunit consisting of four transmembranedomains (Connolly and Wafford, 2004). The physiological agonists of the receptors are gamma

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Hoffmann et al. Pharmacological Activity of Herbal Drugs

aminobutyric acid (GABA) and serotonin (5-HT), respectively.There are many pharmacological similarities between the 5-HT3 and GABAA receptors. For example, the plant-derivedcompound picrotoxin acts as a non-competitive antagonist ofboth receptors (Das and Dillon, 2005), and local anesthetics,such as lidocaine and procaine, can antagonize either receptor(Hara and Sata, 2007; Ueta et al., 2007). A number ofGABAA receptor potentiators or agonists, such as propofol,methohexital, and pentobarbital, are capable of antagonizingthe 5-HT3 receptor (Olsen et al., 1991; Cestari et al., 1996;Barann et al., 2000, 2008). However, while the 5-HT3 receptordisplays a cationic selectivity, activation of the GABAA receptortriggers an influx of Cl−, resulting in the hyperpolarizationof the cell and reduced neuronal excitability. Because of this,the activation of GABAA receptors leads to sedative effects.Therefore, GABAA receptor potentiators and agonists are broadlyused for restlessness and insomnia (Calcaterra and Barrow,2014). For example, the allosteric GABAA receptor potentiatordiazepam is commonly used for psychiatric disorders, includinganxiety and epilepsy (Calcaterra and Barrow, 2014). The 5-HT3receptors are also involved in many pathophysiological processes,such as gastrointestinal motility disorders and the developmentof nausea and vomiting. Therefore, compounds that act on thisreceptor have broad clinical relevance (Doak and Sawynok, 1997;Gershon, 2004; Jeggo et al., 2005; Costedio et al., 2007). Specific5-HT3 receptor antagonists such as ondansetron are mainly usedfor the treatment of nausea for various conditions, includingchemotherapy-induced nausea and vomiting (CINV) and nauseaduring the postoperative phase (PONV; Cubeddu et al., 1994;Gyermek, 1995).

Kampo is a form of traditional Japanese phytomedicineoriginating from traditional Chinese medicine (TCM). Typically,Kampo is administered as a mixture of various herbal drugsthat have complementary physiological activities. Kampo isbroadly used in alternative and complementary medicine and hasalso recently become popular in Western countries. Therefore,there is a general interest in understanding the underlyingpharmacological mechanisms of these herbal drugs, which mayalso help to increase the impact of Kampo in Western medicine.Several previous reports have described the pharmacologicalactions of specific components of single plants (e.g., gingerol fromZingiber officinalis) or complex Kampo preparations consistingof multiple components, such as rikkunshito (Takeda et al.,2008; Tominaga et al., 2011; Herbrechter et al., 2015), onpharmacologically relevant targets, including G-protein-coupledreceptors (GPCRs) and ion channels. During the last decades,screening of the pharmacological activity of plant extracts andsubsequent trials to identify their active ingredients has ledto a plethora of pharmacologically useful substances. Kampomedicine depends on a relatively limited number of 148 well-described mostly plant-derived ingredients (Watanabe et al.,2011). However, only a few attempts were made to establish asystematic and comprehensive screening of the action of all of theimportant Kampo or TCM preparations on specific drug targets,such as ion channels. Nevertheless, such investigations have led tothe identification of new pharmacological tools, as demonstratedby the screening of 50 Chinese herbal plants, which led to the

identification of bisandrographolide as the first natural TRPV4activator (Smith et al., 2006).

Several of the Kampo remedies are used to treat symptomssuch as nausea, emesis, gastrointestinal motility disorders,anxiety, restlessness, and insomnia. Therefore, the pharmaco-logical effects of 121 herbal compounds from Kampo medicinewere analyzed as ethanol tinctures on the heterologouslyexpressed 5-HT3A and GABAA receptors, due to theirinvolvement in the above pathophysiological processes. Weaimed to investigate if there is a correlation between thepharmacological action of the Kampo compounds on thesereceptors and the corresponding medical application. We furthersought to establish an activity ranking and identify the mostpotent tinctures as well as new active ingredients.

MATERIALS AND METHODS

Tinctures and SubstancesThe ethanol tinctures of 121 Kampo remedies were obtainedfrom Dr. Peter Lepke (Kronen Apotheke Wuppertal, Germany).Tinctures were made by extracting the plant material in ethanol(1:5 w/v). The tinctures used in this study are listed inSupplementary Table S1. All of the chemicals were purchasedfrom either Sigma–Aldrich (5-HT hydrochloride, gamma-aminobutyric acid, tannic acid, schizandrin, schizandrin B,leonurine, boldine, berberine chloride, liquiritigenin, hesperetin,kaempferol, andrographolide, linderane, rosmarinic acid, 4-hydroxybenzaldehyd, chlorogenic acid, caffeic acid), Carl Roth(rutin) or PhytoLab (atractylenolide III).

Expression SystemThe expression plasmid pRc/CMV contained cDNA coding forthe human 5-HT3A protein (Invitrogen; Lankiewicz et al., 1998),and psGEM contained cDNA for the α1, β2, and γ2 GABAAreceptor subunits (Sergeeva et al., 2010). cRNAs were preparedusing the AmpliCap T7 high-yield message marker kit (Epicenter,Madison, WI, USA), following the manufacturer’s protocol.Oocytes were obtained as previously described (Sherkheli et al.,2010), and 7–20 ng receptor coding cRNA was injected intothe oocytes using an injection setup from WPI (Nanoliter2000, Micro4). The injected oocytes were then stored in ND96 (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mMMgCl2, 5.0 mM HEPES, pH 7.2, 200 U/ml penicillin, 200 µg/mlstreptomycin) at 14◦C. Electrophysiological experiments wereperformed 1–5 days (5-HT3A) or 2–3 days (GABAA) after thecRNA-injections.

ElectrophysiologyTo investigate the heterologously expressed 5-HT3A and GABAAreceptors, the two-electrode voltage-clamp technique was usedas described previously (Saras et al., 2008). All of the recordingswere performed in a normal frog ringer’s (NFR) buffer (115 mMNaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2) for theGABAA receptor. Currents were recorded at a holding potentialof approximately −60 mV (5-HT3A receptor) or −40 mV(GABAA receptors), using the software Cell Works 6.1.1. (NPI).


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During the measurements, the oocytes were placed in a chamberwith a constant and unidirectional ringer flow, allowing thewashout of the tested substances and tinctures. The ringerflow was interrupted for the application of test substances andtinctures until the maximal response was transcended. To excludesolvent effects, the compounds were tested at the maximalapplied concentration (0.1 Vol.-%). Neither ethanol nor DMSOexhibited a direct activation or modulatory effect on the 5-HT3A and GABAA receptors at the examined concentration (datanot shown). In addition, the action of the tinctures on non-injected oocytes was tested (1:1,000 dilution; n = 3). None of thetinctures exhibited a direct activating effect with currents >15 nA(Supplementary Table S2). The observed effects were negligiblecompared with typical agonist-induced currents (∼2 µA). Thedirect activating effects of the Kampo tinctures were examinedusing a 1:1,000 dilution. The diluted tinctures were applied toXenopus laevis oocytes expressing either the 5-HT3A or α1β2γ2GABAA receptor and compared the evoked responses with theresponse from 5 µM 5-HT or 100 µM GABA, respectively(n = 3–8). For the investigation of the modulatory effects, thetinctures were applied in a 1:1,000-dilutuion witch contains thenative agonists (n = 3–8). Concentrations of 10 µM GABA forthe GABAA receptor and 5 µM 5-HT for the 5-HT3A receptorwere used. To identify the active compounds in tinctures withantagonistic and potentiating effects, the known ingredients ofplants with previously identified potential bioactivity were testedat a concentration of 1 mM. This approach was successfullyused to identify pharmacologically active plant ingredients in aprevious study (Herbrechter et al., 2015).

Data AnalysisThe currents of the tested substances/tinctures were normalizedto the means of the reference compounds 5-HT and GABAfor the 5-HT3A and α1β2γ2 GABAA receptors, respectively.Sigmoidal regression analysis was performed using the 3-or 4-parameter Hill equation (SigmaPlot 8.0., SPSS) to fitconcentration-effect curves and calculate the IC/EC50 values.Deviations are represented by the standard error of the mean(SEM). Significant differences were determined using Student’st-test (Excel 2010, Microsoft), and multiple comparisons werecorrected using the Benjamini–Hochberg-correction (∗p < 0.05;∗∗p < 0.005; ∗∗∗p < 0.0005).

RESULTS

The Effect of Kampo Tinctures on the5-HT3A and GABAA ReceptorsFirst, the direct activating effects of the tinctures wereinvestigated. The tincture of Ligusticum striatum (rhizoma)showed the strongest activation of the 5-HT3A receptor, withmore than 30% of the 5-HT-induced current (Figure 1;Supplementary Table S2). The strongest activator of the GABAAreceptor was Panax ginseng (rhizoma; Ginseng white), with amean current amplitude of more than 40% of the GABA-inducedcurrent. The strongest 12 direct activating tinctures are shown inFigure 1.

Next, the modulatory effects of the tinctures at a 1:1,000dilution was evaluated. In these experiments, agonistconcentrations of 10 µM GABA for the GABAA receptorand 5 µM 5-HT for the 5-HT3A receptor were used. A varietyof tinctures with modulatory action for both receptor typeswere identified. For the 5-HT3A receptor, tinctures withsignificant antagonistic and potentiating effects were identified(13 tinctures with potentiating effects and 22 tinctures withinhibitory potential). The strongest potentiation was observedwith Chaenomeles speciosa (fructus), which potentiated the5-HT-induced response with only 37% (Figure 2). In contrast,eight tinctures [Tetradium ruticarpum (fructus), Magnoliabiondii (flos), Lonicera japonica (caulis), Paeonia × suffruticosa(cortex), Coptis chinensis (radix), Epimedium brevicornum(herba), Leonurus japonicus (herba), and Lindera aggregata(radix)) exceeded 50% inhibition of the receptor, with amaximal inhibition of more than 98% by the Lindera aggregata(radix) tincture. In addition, the GABAA receptors weresignificantly potentiated by 14 tinctures and inhibited by 24tinctures. The Syzygium aromaticum (flos), Clematis armandii(caulis), Magnolia officinalis (cortex), Mentha canadensis(herba), Scutellaria baicalensis (radix), and Panax ginseng(rhizoma; Ginseng red) tinctures exhibited the strongestpotentiation. In particular, Ginseng red potentiated up to 135%(Figure 3). The most effective inhibitory tinctures were Salviamiltiorrhiza (radix), Caesalpinia sappan (lignum), Terminaliachebula (fructus), Gentiana macrophylla (radix), Saposhnikoviadivaricata (radix), and Schisandra chinensis (fructus). Theinhibition observed by the Salvia miltiorrhiza (radix) tinctureexceeded an inhibition of 80% of the GABA-induced currents.

Investigation of Established Ingredientsin the Active Kampo TincturesFor several of the active tinctures, the active ingredients havealready been identified. However, for many of the Kampo herbsthat have been shown to act on the 5-HT3A or GABAA receptor,the active ingredients are unknown. The investigated substancesand their related plants are listed in Table 1 and SupplementaryTable S3.

The modulatory effects of these substances on both receptorsare shown in Figure 4. Overall, alkaloid compounds werefound to be the most effective antagonists. In particular,boldine [Lindera aggregata (radix)] and leonurine [Leonurusjaponicus (herba)] each possessed a higher efficacy for the 5-HT3A receptor than the GABAA receptor. These compoundswere the most potent 5-HT3A receptor antagonists in thescreening (Figure 4). The alkaloid berberine inhibited bothreceptor types at approximately 60%. Another identified 5-HT3A receptor antagonist is the flavonoid (-)-liquiritigenin(Herbrechter et al., 2015), which showed nearly no effect onGABAA receptors in this screen. All of the tested flavonoidsinhibited 5-HT3A receptors with higher efficacy than GABAAreceptors. However, many of the substances showed inhibitorypotential rather than potentiating effects on the receptors, withthe exception of schizandrin B, which slightly potentiated theGABAA receptor. Within the phenolic compound category,



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FIGURE 1 | The strongest 12 direct activating tinctures for the 5-HT3A (A) and GABAA receptors (B). The 121 tinctures were made from Kampo remediesvia ethanol extraction (see section Tinctures and substances). A 1:1,000-dilution was applied to the oocytes and compared with agonist induced currents (5 µM5-HT, 100 µM GABA). Error bars represent the SEM. Statistical significance was calculated based on the current evoked by ethanol (0.1 Vol.-%; ∗p < 0.05,∗∗p < 0.005; n = 3–5).

tannic acid was identified as an antagonist of both receptor types,with an inhibition rate of more than 80%. Andrographolide andthe lignane schizandrin were identified as weak GABAA receptorantagonists.

Concentration-Effect Curves of theIdentified Ingredients with PotentialPharmaceutical ActionTo quantify the antagonism of the identified active ingredients,concentration-effect curves were generated (Figure 5;Supplementary Table S4).

For the 5-HT3A receptor, boldine (IC50 = 0.53 ± 0.15 µM)and leonurine (IC50 = 2.17 ± 0.15 µM) were more potentinhibitors than tannic acid (IC50 = 48.2 ± 4.1 µM) andschizandrin (IC50 = 137 ± 22 µM). These four substanceshad approximately equal IC50 values for the GABAAreceptor (∼100 µM, Supplementary Table S4), with theexception of schizandrin (IC50 > 600 µM). Andrographolide(IC50 = 66.1 ± 26.8 µM) was found to be the most potentGABAA receptor antagonist. However, it also acted as a partialantagonist of the GABAA receptor, as even a concentration of3 mM reduced the GABA-evoked current to 30% of the GABA



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FIGURE 2 | The modulatory action of the strongest inhibitory and potentiating Kampo tinctures on the 5-HT3A receptor. (A) The tinctures with the 10strongest antagonistic (left) and potentiating (right) effects are shown (5 µM 5-HT). Bars exceeding the dotted line represent potentiating effects. Statisticalsignificance was calculated based on the reference solution containing 0.1 Vol.-% ethanol (equal to the max. ethanol concentration in the tinctures; ∗p < 0.05,∗∗p < 0.005, ∗∗∗p < 0.0005; n = 3–8). (B) Original registrations of the modulation observed by Leonurus japonicus (herba) and Cannabis sativa (semen) using a1:1,000 dilution are shown. The effects were reversible following a 150-s washout with ringer’s solution.

reference (Figure 5B). Leonurine shifted the EC50 for 5-HTat the 5-HT3A receptor from 2.5 µM determined in parallelunder the same experimental conditions in our laboratory(Herbrechter et al., 2015) to 14.8 µM. While 30 µM leonurinealmost completely blocked the evoked current at lower agonistconcentrations, it was nearly ineffective at 5-HT concentrations≥100 µM, suggesting a competitive antagonism of leonurine atthe 5-HT3A receptors.

DISCUSSION

In this screening, two classical drug targets, the 5-HT3and GABAA receptors, were investigated. Several Kamporemedies have anxiolytic, sedative, antiemetic, or digestive effects,suggesting that these receptors may be the pharmacological

targets of Kampo compounds. Therefore, 121 Kampo remedieswere screened as ethanol extracts on heterologously expressedα1β2γ2 GABAA and homomeric 5-HT3A receptors, using thetwo-electrode voltage-clamp technique. As a result, severalremedies that can modulate the 5-HT- or GABA-inducedcurrents were found. An activity ranking of the activity of eachplant preparation on these receptors was established. By testingsingle substances that are present in the active plant extracts,several new 5-HT3A blockers and GABAA receptor modulatorswere identified.

Pharmacologically Active KampoRemedies: 5-HT3A ReceptorDuring the screening for compounds that are pharmacologicallyactive at the 5-HT3A receptor, a number of remedies with



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TABLE 1 | Investigated ingredients and their respective plants.

Substance Kampo remedy Reference

4-hydroxybenzaldehyd Lonicera japonica (caulis) Wang et al., 2013

andrographolide Andrographis paniculata (herba) Song et al., 2013

Atractylenolid III Atractylodes lancea (rhizoma)Atractylodis macrocephala(rhizoma)

Shao et al., 2014

Berberine Coptis chinensis (radix) Lin et al., 2004

Boldine Lindera aggregata (radix) Han et al., 2008

Chlorogenic acid Inula helenium (radix)Lonicera japonica (caulis)

Eberhard, 2003;Wang et al., 2014

Caffeic acid Inula helenium (radix) Wang et al., 2014

Hesperetin Citrus × aurantium (fructus)Citrus trifoliata (fructus)Citrus reticulata (pericarpium,vir.)Citrus reticulata (pericarpium)

Zhao et al., 2015

Kaempferol Houttuynia cordata (herba) Lin et al., 2013

Leonurine Leonurus japonicus (herba) Chen and Kwan,2001

Linderane Lindera aggregata (radix) Li et al., 2002

Liquiritigenin Glycyrrhiza uralensis (radix) Rauchensteineret al., 2005; Kondoet al., 2007

Rosmarinic acid Salvia miltiorrhiza (radix) Adams et al., 2006;Huang et al., 2008

Rutin Citrus × aurantium (fructus)Citrus trifoliata (fructus)Citrus reticulata (pericarpium,vir.)Citrus reticulata (pericarpium)Morus alba (folia)

Hunyadi et al.,2012; Zhao et al.,2015

Schizandrin B Schisandra chinensis (fructus) Pan et al., 2008

Schizandrin Schisandra chinensis (fructus) Panossian andWikman, 2010

Tannic acid Syzygium aromaticum (flos) Bhowmik et al.,2012

antagonistic properties were identified. Several of these remedies,such as ginger [Zingiber officinalis (rhizoma)], have well-described antiemetic effects and are used for the treatment ofnausea and gastrointestinal disorders (Ernst and Pittler, 2000;Haniadka et al., 2012; Ding et al., 2013). During our screen,rhizoma from Zingiber off. sicc. and Zingiber off. vir. exhibitedantagonistic effects, and the effect of Zingiber off. sicc. (desiccated)was superior to that observed with the tincture of fresh rhizomafrom Zingiber off. vir. This increased inhibition may be due tothe manufacturing process of the tinctures, as a higher amountof dry matter was used for the Zingiber off. sicc. tincture, andthis may have resulted in an increase in the concentration ofthe active ingredients within the tincture. Vanilloids from thegingerol and shogaol group of compounds, pungent substancesof ginger, are well-known 5-HT3A receptor antagonists. Thismay explain, in part, the observed inhibition of 5-HT-inducedcurrents that have been reported previously (Walstab et al.,2013; Ziemba et al., 2015). In addition, terpenes within theessential oil of ginger, including geraniol, citronellol, linalool andgalanolactone, were shown to antagonize the 5-HT3A receptor(Huang et al., 1991; Ziemba et al., 2015). The antagonistic

potential of Panax ginseng (radix) tinctures may be mediated byginsenosides, which act in the pore region of the 5-HT3A receptor,and its antiemetic properties have been previously described(Min et al., 2003; Kim et al., 2005; Lee et al., 2007). The Magnoliabiondii (flos), Mentha canadensis (herba), Glycyrrhiza uralensis(radix) and Syzygium aromaticum (flos) tinctures, which are alsoreported to have antiemetic effects (World Health Organization,2005; Bhowmik et al., 2012; Herbrechter et al., 2015), wereamong the top 20 inhibitory tinctures in the screening. Syzygiumaromaticum (flos) contains the phenolic compounds eugenol,gallic acid, and tannins, as well as the flavonoid quercetin(Atawodi et al., 2011; Bhowmik et al., 2012). Instead of eugenol(the main flavoring ingredient in Syzygium aromaticum), whichshows a negligible level of antagonism (Ziemba et al., 2015),tannic acid inhibits 5-HT-evoked currents with an IC50 valueof approximately 50 µM (Figure 5). In addition to tannic acid,quercetin may contribute to the antagonistic effect of the clovetincture (Lee et al., 2008). Glycyrrhiza uralensis (radix) containsa structurally related flavonoid, liquiritigenin, which has beenshown to antagonize the 5-HT3A receptor in a previous study.This finding may help us to understand the antagonistic effect ofthis tincture (Herbrechter et al., 2015). Another flavonoid fromlicorice, named glabridin, was identified as a partial antagonistof the 5-HT3A-receptor and a potentiator of GABAA-receptors(Herbrechter et al., 2015; Hoffmann et al., 2016). The antagonisticeffect of Mentha canadensis (herba) may be explained throughthe action of terpenes, which are known to inhibit the 5-HT3A receptor. Menthol, a terpene from the essential oil ofMentha canadensis (herba), was recently characterized as a 5-HT3 receptor antagonist (Walstab et al., 2014). In addition, Coptischinensis rhizomes (Tjong et al., 2011) and Lonicera japonica(flos; Jung et al., 2014) may be useful for the treatment ofgastrointestinal disorders through the action 5-HT3 receptors, asdiscussed below.

The most potent inhibitory effects were observed for thetinctures of Lindera aggregata (radix) and Leonurus japonicus(herba; Figure 2; Supplementary Table S2) for which noconnection to gastrointestinal disorders is described in theliterature. By screening known constituents of these plants,boldine (Lindera) and leonurine (Leonurus) are found as potentinhibitors of the 5-HT3A receptor with an IC50 between 0.5and 2.3 µM (Figures 5 and 6). Boldine, which has been shownto ameliorate chemotherapy-induced nausea and vomiting aswell as symptoms from irritable bowel syndrome, is an alkaloidfrom the aporphine class. Boldine has been previously shownto antagonize the 5-HT3A receptor in a competitive mannerin a luminescence-based cell assay (Walstab et al., 2014). Thecontribution of further ingredients, such as the related alkaloidsnorboldine, reticuline, and linderegatine, to the antagonisticeffect of the Lindera tincture is likely (Han et al., 2008).Leonurine from Leonurus was shown to affect cardiac functionvia creatine kinase inhibition (Wang et al., 2004) and to alsoreduce platelet aggregation (Lin et al., 2007). These effects arein accordance with some of the traditional uses of Leonurus.The antagonistic effects of Leonurus and the identification ofleonurine as a potent competitive 5-HT3A receptor antagonist arereported here for the first time. Therefore, this study reveals new



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FIGURE 3 | The modulatory action of the strongest inhibitory and potentiating Kampo tinctures on the GABAA receptors. (A) The tinctures with the 10strongest antagonistic (left) and potentiating (right) effects are shown (10 µM GABA). Statistical significance was calculated based on the reference solutioncontaining 0.1 Vol.-% ethanol (equal to the max. ethanol concentration in the tinctures; ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005; n = 3–8). (B) Original registrations ofthe modulation observed via Saposhnikovia divaricata (herba) and Magnolia officinalis (cortex) with a 1:1,000 dilution are shown. The effects were reversible followinga 150-s washout with ringer’s solution.

insights into the pharmacological action of Leonurus. However,the traditional application of Leonurus is not correlated withthe pharmacology of the 5-HT3 receptor (Shang et al., 2014).Therefore, further studies examining if Leonurus can antagonizethe 5-HT3A receptor in vivo are needed.

Pharmacologically Relevant KampoRemedies: GABAA ReceptorThe GABAA receptors are inhibitory receptors and thereforereduce neuronal excitability. A variety of disorders are

attributable to an imbalance of the GABAergic system.Therefore, benzodiazepines, such as diazepam, potentiatethe GABA-induced responses and are used to treat disorderssuch as insomnia, restlessness, anxiety, and epilepsy (Calcaterraand Barrow, 2014).

In this screening, the potentiating activity of the tincturescorrelated well with the medical use of the plants. For manyof the top 10 potentiation tinctures, the active ingredients hadbeen previously identified. The best potentiator, Panax ginseng(rhizoma; Ginseng red) is likely one of the most popular herbal



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FIGURE 4 | Modulatory effects of the investigated ingredients on the 5-HT3A and GABAA receptors. (A) The bars represent the normalized currentamplitude for the modulation of the 5-HT3A (gray bars) and GABAA (white bars) receptors. With the exception of schizandrin B, which was tested at 100 µM, all ofthe compounds were evaluated at a concentration of 1 mM. Bars exceeding the dotted line represent substances with potentiating effects. The substances arearranged based on their affiliation to classes of plant metabolites, as indicated by the vertical lines. Statistical differences were calculated based on the referenceagonist, which contained an equal concentration of the solvent used to dissolve the test compounds (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005; n = 4–7). (B) Originalregistrations of the inhibition of the 5-HT3A receptor by tannic acid and leonurine. (C) Original registrations of the inhibition of the GABAA receptor by tannic acid andleonurine.

drugs. It is traditionally used for multiple purposes, includingthe treatment of anxiety and insomnia (Kim et al., 2005;Xiang et al., 2008; Lee et al., 2013). The ginsenosides (steroidglycosides) present in this compound may be responsible forthe potentiation and hence the sedative and anxiolytic effects ofginseng, due to their potentiating effect on the GABAA receptor.The ginsenosides Rg3 and Rc have been shown to potentiate theheterologously expressed GABAA receptor, and ginsenoside Rc

also possesses agonistic properties toward the GABAA receptor(Choi et al., 2003; Lee et al., 2013). This may explain theobserved direct activating effect of the extract (Figure 1). TwoPanax ginseng (radix) tinctures were also tested, which differ intheir manufacturing process. Whereas Ginseng red is desiccateddirectly after harvesting, Ginseng white is peeled and whitenedprior to the desiccation process. Despite these manufacturingdifferences, both compounds were of the best nine potentiating



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FIGURE 5 | Concentration-inhibition curves of select plant ingredients for the 5-HT3A (A) and GABAA (B) receptor, as well as the original registrationsof leonurine for the 5-HT3A receptor (C) and the 5-HT concentration-effect curve in the presence of leonurine (D). (A,B) The agonist concentrations usedin the experiment were 5 µM (5-HT) or 3 µM (GABA). Error bars represent the SEM (A: n = 5–7, B: n = 4–7). Andrographolide acts as a partial antagonist of theGABAA receptor. (C) Overlayed 5-HT3A receptor responses, modulated by different concentrations of leonurine (0.1–10 µM) are shown. (D) The concentration-effectcurve of 5-HT at the 5-HT3A receptor in the presence of leonurine (30 µM) is shown. The determined EC50 value was found to be 14.8 µM 5-HT (n = 4).

tinctures. However, Ginseng red is superior to Ginseng whitedue to its increased potentiation (135% instead of 30%; Figure 3;Supplementary Table S2).

The Scutellaria baicalensis (radix) tincture potentiated theGABAA receptor at a similar level to Panax ginseng (rhizoma;Ginseng red; Figure 3; Supplementary Table S2), and itwas also previously linked to GABAA receptor-related effects.Scutellaria baicalensis (radix) possesses anticonvulsant, sedativeand anxiolytic effects, which may be explained by the action ofthe flavone wogonin (Hui et al., 2002; Park et al., 2007). Wogoninwas identified as a ligand for the benzodiazepine site of theGABAA receptor and was shown to potentiate GABAA receptorsin electrophysiological assays (Hui et al., 2002). The observed

anxiolytic effects of wogonin in mice were similar to that ofdiazepam, emphasizing the potency of wogonin (Hui et al., 2002).The Mentha canadensis (herba) and Magnolia officinalis (cortex)tinctures also exhibited a potentiating effect in the screening(Figure 3; Supplementary Table S2). The active components ofMagnolia officinalis (cortex) are the lignans magnolol and itsisomer honokiol, whose potentiation of the GABAA receptor isaccountable for the anticonvulsant, antidepressive, and anxiolyticeffects of Magnolia bark (Taferner et al., 2011; Alexeev et al.,2012). The potentiation of the GABAA receptor by the Menthatincture likely occurs due to the action of menthol and thestructurally related monoterpenoids, which were shown topotentiate GABAA receptors in a previous study (Hall et al.,



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FIGURE 6 | Molecular structures of the alkaloids leonurine, boldine,and tannic acid.

2004). In addition, extracts of Morus alba displayed anxiolyticeffects under different experimental paradigms, suggesting thatthis compound may also act on the GABAA receptor (Yadavet al., 2008). Our results support this idea, as Morus alba (folia)extracts belonged to the top 10 most potent GABAA potentiators.However, the active component in Morus alba (folia) extract isstill unknown.

The sixth-strongest potentiation was observed by theclove tincture [Syzygium aromaticum (flos)], which has anti-inflammatory, antimicrobial, antioxidant, and anesthetic effects(Chaieb et al., 2007). In vivo, eugenol, the main ingredient ofthe clove tincture, exhibits a sedative effect in mice and rats(Guenette et al., 2006; Sharma et al., 2012) and has also beenshown to potentiate the effects of the GABAA receptor in vitro(Aoshima and Hamamoto, 1999). Other potentiators were alsoidentified, including carvacrol and thymol (Priestley et al.,2003; Kessler et al., 2014). In addition to eugenol, Syzygiumaromaticum (flos) contains carvacrol and thymol (Priestley et al.,2003; Kessler et al., 2014), and all three are potentiators of theGABAA receptor. The mutual action of these compounds mayexplain the sedative effects of Syzygium aromaticum (flos).

Antagonism of the GABAA receptor is typically accompaniedby stimulating effects at low doses but can cause seizuresand anxiety at higher concentrations. Kampo remedies withantagonistic effects were detected, including the tinctures ofSalvia miltiorrhiza (radix), Terminalia chebula (fructus), andSchisandra chinensis (fructus; Figure 3; Supplementary Table S2).However, we could not find an anxiogenic or seizure-inducingadverse side effect for these antagonistic tinctures in theliterature. The Schisandra chinensis (fructus) tincture, whichinhibited 50% of the GABA-induced current at a 1:1,000 dilution(Figure 3; Supplementary Table S2), possesses side effects such

as restlessness and tension, when given at high doses (Eberhard,2003). These effects may be explained, at least in part, by itsantagonism of the GABA receptor. Nevertheless, Schisandrachinensis (fructus) is traditionally used for its hepatoprotectiveand sedative effects; the latter is based on the influence ofthe GABAergic and serotonergic systems, as the levels of theirrespective neurotransmitters can be altered in the brain (Ipet al., 1996; Wei et al., 2013; Zhang et al., 2014). The lignanoidschizandrin was identified as a weak inhibitor of the GABAAreceptor (Figures 4 and 5; Supplementary Table S4), presumablyacting in a non-competitive manner (Supplementary Figure S1).However, its contribution to the effect of the tincture remainshypothetically, with regard to the low potency.

The Andrographis paniculata (herba) tincture exhibited aweak antagonism (Supplementary Table S2). However, thetested labdane diterpenoid andrographolide, which possessesantiphlogistic, anticancer, and neuroprotective effects (Chanet al., 2010; Chun et al., 2010; Kou et al., 2014; Linet al., 2014), is a partial antagonist of the GABAA receptor(Figure 5; Supplementary Table S4). Moreover, the actionof andrographolide appears to be non-competitive in nature(Supplementary Figure S2). The andrographolide content ofthe A. paniculata leaves was approximately 1% of the dryweight (Chao and Lin, 2010). Hence, the concentration in theapplied tincture can be approximated (≈6 µM). Therefore, theantagonistic effect of the tincture is not solely due to the action ofandrographolide.

The Salvia miltiorrhiza (radix) tincture exhibited the strongestantagonistic effect on the GABAA receptor, exceeding a blockingeffect of 80% (Figure 3; Supplementary Table S2). This compoundis traditionally used for the treatment of cardiovascular diseasesand ischemia, and it inhibits platelet aggregation via the actionof tanshinones (Han et al., 2008). One specific tanshinone,miltirone, was identified as a ligand at the benzodiazepinebinding site of the GABAA receptor in the CNS, based onligand binding assays (Lee et al., 1991). Nevertheless, miltironefailed to modulate the GABA-induced current of the GABAAreceptor in a recombinant expression system (Xenopus oocytes)as well as in cultured rat hippocampal pyramidal cells (Mostallinoet al., 2004). Hence, the involvement of miltirone in theobserved inhibition of GABAA receptor responses by the Salviamiltiorrhiza tincture seems unlikely. Additionally, rosmarinicacid, a monoterpenoid from Salvia miltiorrhiza (Adams et al.,2006), only showed a slight inhibition of the GABAA receptor inthe screening (Figure 4; Supplementary Table S3) and thereforedoes not account for the strong antagonistic effect of the tincture.

Salvia miltiorrhiza and Astragalus propinquus are compoundsin a two-ingredient intermixture called myelophil. This mixturehas been shown to reduce chemotherapy-correlated adverseside effects, including myelosuppression and anemia, from thecytostatic drug fluorouracil (Shin et al., 2008) and possessesno described side effects itself (Jung et al., 2009). Furthermore,myelophil reduced fatigue in patients suffering from chronicfatigue (Cho et al., 2009) and exhibited anti-amnesic effectsin scopolamine-treated mice (Lee et al., 2014). Even if thelatter is presumably caused by the increased expression ofERKs and mAChR1 (Lee et al., 2014), a contribution of the



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inhibition of Salvia miltiorrhiza on the GABAA receptor to theanti-fatigue effects of myelophil is likely, due to the increasedneuronal excitability resulting from GABAA receptor inhibition.In contrast to Salvia miltiorrhiza, Astragalus propinquus didnot affect the GABAA receptor in this study (SupplementaryTable S2).

CONCLUSION

We believe that a broad screening of the ethanol tincturesof herbal remedies via electrophysiological assays is a reliablemethod to obtain an overview of the pharmacological action ofthese compounds on clinically relevant receptors. In this study,a variety of tinctures were detected, whose pharmacologicalactions on the investigated receptors are in agreement withtheir traditional uses and physiological effects. For example,the antiemetic remedies Zingiber officinalis (rhizoma), Panaxginseng (radix), and Syzygium aromaticum (flos) were identifiedas antagonists of the 5-HT3A receptor, and sedative drugs suchas Panax ginseng (radix), Scutellaria baicalensis (radix), andMentha canadensis (herba) were found as GABAA receptorpotentiating tinctures. Furthermore, Lindera aggregata (radix)and Leonurus japonicus (herba) are identified as potent 5-HT3Areceptor antagonists. Boldin was recently identified as a potent5-HT3A receptor blocker (Walstab et al., 2014) and is likely theactive component of Lindera aggregata (radix). In this study,leonurine was identified as a new natural 5-HT3A receptorantagonist. It inhibited the 5-HT3A receptor with a similarpotency to boldine. Furthermore, tinctures that antagonize theGABAA receptor, were detected. E.g., Salvia miltiorrhiza (radix),which is part of a Kampo remedy called myelophil, blocksGABAA receptor responses with high efficacy. Myelophil waspreviously shown to ameliorate fatigue symptoms. Therefore,we hypothesized that Salvia miltiorrhiza (radix) may contributeto the anti-fatigue effects of myelophil due to the increased

neuronal excitability accompanied by the antagonism of theGABAA receptor. Hence, electrophysiological screens are ahelpful tool to understand the physiological effects of herbaldrugs and enable the identification of new bioactive compounds,such as leonurine, for the development of new pharmaceuticsor the more precise application of herbal remedies. Thedevelopment of new pharmaceutical drugs out of the identifiedactive ingredients of these herbs is a further possibility. Theknowledge regarding the medicinal benefit of Kampo remedieshas continued to evolve for centuries. This study can be thoughtof as an attempt to “translate” this traditional system into acontemporary, pharmacological system. Here, two classical drugtargets were investigated, the 5-HT3 and GABAA receptors.However, there is still little knowledge with regard to thepharmacological action of many Kampo remedies on otherclinically relevant targets, and therefore, there is great potentialfor future research.

AUTHOR CONTRIBUTIONS

KH, HH, MW, and GG conceived and designed the experiments;KH, RH, PZ, and LB performed the experiments; PL contributedmaterials; KH, RH, and GG wrote the paper.

ACKNOWLEDGMENT

We would like to thank C. Wecker, A. Hupperich, and U. Müllerfor the assistance provided.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: http://journal.frontiersin.org/article/10.3389/fphar.2016.00219

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

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Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A ReceptorsIntroductionMaterials And MethodsTinctures and SubstancesExpression SystemElectrophysiologyData Analysis

ResultsThe Effect of Kampo Tinctures on the 5-HT3A and GABAA ReceptorsInvestigation of Established Ingredients in the Active Kampo TincturesConcentration-Effect Curves of the Identified Ingredients with Potential Pharmaceutical Action

DiscussionPharmacologically Active Kampo Remedies: 5-HT3A ReceptorPharmacologically Relevant Kampo Remedies: GABAA Receptor

ConclusionAuthor ContributionsAcknowledgmentSupplementary MaterialReferences

kampo medicine: evaluation of the pharmacological activity of 121 herbal...

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